Analysis of Semen Parameters, Sperm Activity, and Fertility of Somatic Cell Cloned Hanwoo Bulls

체세포 복제 한우 수소의 정액 성상, 정자의 활동성 및 수정 능력 분석

  • Bae, Seong-Hoon (Animal Biotechnology Division, National Institute of Animal Science, RDA) ;
  • Hwang, Seong-Soo (Animal Biotechnology Division, National Institute of Animal Science, RDA) ;
  • Yang, Byong-Chul (Animal Biotechnology Division, National Institute of Animal Science, RDA) ;
  • Go, Yeoung-Kyu (Animal Biotechnology Division, National Institute of Animal Science, RDA) ;
  • Kim, Dong-Hoon (Animal Biotechnology Division, National Institute of Animal Science, RDA) ;
  • Im, Gi-Sun (Animal Biotechnology Division, National Institute of Animal Science, RDA) ;
  • Choi, Hwa-Sik (Department of Clinical Laboratory Science, Shin Heong College) ;
  • Jin, Dong-Il (Division of Animal Science & Resources, Chungnam National University) ;
  • Yang, Boh-Suk (Animal Biotechnology Division, National Institute of Animal Science, RDA) ;
  • Seong, Hwan-Hoo (Animal Biotechnology Division, National Institute of Animal Science, RDA)
  • 배성훈 (농촌진흥청 축산과학원 응용생명공학과) ;
  • 황성수 (농촌진흥청 축산과학원 응용생명공학과) ;
  • 양병철 (농촌진흥청 축산과학원 응용생명공학과) ;
  • 고응규 (농촌진흥청 축산과학원 응용생명공학과) ;
  • 김동훈 (농촌진흥청 축산과학원 응용생명공학과) ;
  • 임기순 (농촌진흥청 축산과학원 응용생명공학과) ;
  • 최화식 (신흥대학교 임상병리학과) ;
  • 진동일 (충남대학교 동물자원학부) ;
  • 양보석 (농촌진흥청 축산과학원 응용생명공학과) ;
  • 성환후 (농촌진흥청 축산과학원 응용생명공학과)
  • Published : 2007.09.30

Abstract

This study was performed to investigate the reproductive characteristics of the cloned Hanwoo bulls produced by SCNT. The semen ejaculated from the cloned bulls (C-38 and C-39) and normal Hanwoo bull was properly measured the volume, the number of sperm, and the viability of frozen-thawed sperm. The sperm activity was analyzed using computer assisted sperm analysis (CASA). To analyze fertilizing ability of the cloned bulls, in vitro fertilization and artificial insemination were performed using the frozen-thawed semen. There were no differences in semen volume, sperm concentration, and the viability of frozen-thawed sperm between cloned bulls and normal bull. The difference was statistically significant in total motility, curvilinear velocity (VCL), straight-line velocity (VSL), and average-path velocity (VAP) of both cloned bulls compared to those of normal Hanwoo bull, respectively (p<0.05). The cleavage and blastocyst development rate were not different between the groups. five cloned cows were artificially inseminated using the frozen-thawed semen of C-38, two of them became pregnant. Two second generation calves (one male and one female) were produced. Based on these results, the cloned Hanwoo bulls showed normal reproductive abilities of semen parameters and sperm activity to their comparators and produced cloned calves, although there are some individual differences on the parameters.

본 연구는 체세포를 이용하여 생산된 복제 한우 수소의 번식능력을 검토하기 위해 실시하였다. 복제 한우 수소(C-38 및 C-39) 또는 일반 한우 종모우로부터 정액을 채취하여 정자의 수 및 동결 전 후의 생존성 등을 살펴보았으며, 정자의 운동성 등은 computer assisted sperm analysis(CASA)를 이용하여 측정하였다. 또한, 이들의 수정 능력을 확인하기 위하여 체외수정과 인공수정을 각각 실시하였다. 정액 성상에서는 복제 수소들과 일반 종모우 간에 정액의 양, 정자의 농도 및 동결융해 후의 생존성 등에서 차이가 나타나지 않았다. CASA를 이용한 분석에서 운동성, 곡선 운동 속도(VCL), 직선운동 속도(VSL) 및 평균 진행 속도(VAP) 등은 복제 수소의 정액이 일반 종모우의 정액에 비하여 유의적으로 높았다 (p<0.05).체외수정에 따른 수정란의 분화율 및 배반포로의 발달율은 복제 수소와 일반종모우 간에 차이가 나타나지 않았다. 복제소 정액(C-38)을 이용하여 인공수정을 한 5두의 체세포 복제 대리모에서 암 수 각각 한 두씩의 건강한 복제 후대 송아지 2두를 생산하였다. 이상의 결과를 종합하여 보면, 실험에 공시된 복제 수소 개체간의 차이가 나타나기는 하였지만, 복제 수소는 정액 성상과 정자의 운동성 등에서 일반 종모우와 차이가 없었으며, 또한 인공수정을 통해 송아지를 생산함으로써 정상적인 번식능력이 있음을 확인하였다.

Keywords

References

  1. Bailey JL, Bilodeau JF, Cormier N (2000): Semen cryopreservation in domestic animals: a damaging and capacitating phenomenon. J Androl 21:1-7
  2. Brito LFC, Silva AEDF, Rodrigues LH, Vieira FV, Deragon LAG, Kastelic JP (2002): Effect of age and genetic group on characteristics of the scrotum, testes testicular vascular cones, and on sperm production and semen quality in AI bulls in Brazil. Theriogenology 58:1175-1186 https://doi.org/10.1016/S0093-691X(02)00921-4
  3. Campbell KH, McWhir J, Ritchie WA, Wilmut I (1996): Sheep cloned by nuclear transfer from cultured cell line. Nature 380:64-66 https://doi.org/10.1038/380064a0
  4. Cibelli JB, Stice SL, Golueke PJ, Kane JJ, Jerry J, Blackwell C, Ponce de Leon FA, Robl JM (1998): Cloned transgenic calves produced from nonquiescent fetal fibroblasts. Science 280: 1256-1258 https://doi.org/10.1126/science.280.5367.1256
  5. Farell PB, Presicce GA, Brockett CC, Foote RH (1998): Quantification of bull sperm characteristics measured by computer assisted sperm analysis (CASA) and the relationship to fertility. Theriogenology 49:871-879 https://doi.org/10.1016/S0093-691X(98)00036-3
  6. Gillan L, Evans G, Maxwell WM (2005): Flow cytometric evaluation of sperm parameters in relation to fertility potential. Theriogenology 63:445-457 https://doi.org/10.1016/j.theriogenology.2004.09.024
  7. Hafez ESE (1993): Preservation and cryopreservation of gametes and embryos. In: Hafez ESE, ed. Reproduction in Farm Animals. Philadelphia, PA: Lea & Febiger; pp. 503-525
  8. Hallap T, Jaakma U, Rodriguez-Martinez H (2006): Changes in semen quality in Estonian Holstein AI bulls at 3, 5 and 7 years of age. Reprod Domest Anim 41:214-218 https://doi.org/10.1111/j.1439-0531.2006.00682.x
  9. Heyman Y, Richard C, Rodriguez-Martinez H, Lazzari G, Chavatte-Palmer P, Vignon X, Galli C (2004): Zootechnical Performance of Cloned Cattle and Offspring: Preliminary Results. Cloning Stem Cells 6:111-120 https://doi.org/10.1089/1536230041372364
  10. Kato Y, Tani T, Sotomaru Y, Kurokawa K, Kato J, Doguchi H, Yasue H, Tsunoda Y (1998): Eight calves cloned from somatic cells of a single adult. Science 282:2095-2098 https://doi.org/10.1126/science.282.5396.2095
  11. Kubota C, Yamakuchi H, Todoroki J, Mizoshita K, Tabara N, Barber M, Yang X (2000): Six cloned calves produced from adult fibroblast cells after long-term culture. Proc. Natl Acad Sci USA 97:990-995
  12. Leibo SP, Bradley L (1999): The male gamete from basic science to clinical applications. In: Gagnon C, editor. Vienna, IL: Cache River Press 501-516
  13. Lessard C, Masseau I, Bilodeau JF, Kroetsch T, Twagiramungu H, Bailey JL, Leclerc P, Sullivan R (2003): Semen characteristics of genetically identical quadruplet bulls, Theriogenology 59:1865-1877 https://doi.org/10.1016/S0093-691X(02)01256-6
  14. Martinez-Pastor F, Diaz-Corujo AR, Anel E, Herraez P, Anel L, de Paz P (2005a): Postmortem time and season alter subpopulation characteristics of Iberian red deer epididymal sperm. Theriogenology 64:958-974 https://doi.org/10.1016/j.theriogenology.2005.01.003
  15. Martinez-Pastor F, Guerra C, Kaabi M, Diaz AR, Anel E, Herraez P, de Paz P, Anel L (2005b): Decay of sperm obtained from epididymides of wild ruminants depending on postmortem time. Theriogenology 63:24-40 https://doi.org/10.1016/j.theriogenology.2004.03.003
  16. Park CG, Kim SW, Lee PY, Han JH, Lee HG, Byun SJ, Yang BS, Lee CH, Lee HT, Chang WK, Park JK, (2006): Sperm Fertility of Transgenic Boar Harboring hEPO Gene is Decreased. Reprod Dev Biol 30: 27-34
  17. Polejaeva lA, Chen SH, Vaught TD, Page RL, Mullins J, Ball S, Dai Y, Boone J, Walker S, Ayares DL, Colman A, Campbell KH (2000): Cloned pigs produced by nuclear transfer from adult somatic cells. Nature 407:86-90 https://doi.org/10.1038/35024082
  18. Shiga K, Umeki H, Shimura H, Fujita T, Watanabe S, Nagai T (2005): Growth and fertility of bulls cloned from the somatic cells of an aged and infertile bull. Theriogenology 64:334-343 https://doi.org/10.1016/j.theriogenology.2004.12.002
  19. Tecirlioglu RT, Cooney MA, Korfiatis NA, Hodgson R, Williamson M, Downie S, Galloway DB, French AJ (2006): Semen and reproductive profiles of genetically identical cloned bulls. Theriogenology 65: 1783-1799 https://doi.org/10.1016/j.theriogenology.2005.09.033
  20. Verstegen J, Iguer-Ouada M, Onclin K, (2002): Computer assisted semen analyzers in andrology research and veterinary practice. Theriogenology 57: 149-179 https://doi.org/10.1016/S0093-691X(01)00664-1
  21. Wakayama T, Perry AC, Zuccotti M, Johnson KR, Yanagimachi R (1998): Full-term development of mice from enucleated ooeytes injected with cumulus cell nuclei. Nature 394:369-374 https://doi.org/10.1038/28615
  22. Waston PF (1995): Recent developments and concepts in the cryopreservation of spermatozoa and the assessment of their post-thawing function. Reprod Fertil Dev 7:871-891 https://doi.org/10.1071/RD9950871
  23. Waston PF (2000): The causes of reduced fertility with cryopreserved semen. Anim Reprod Sci 61: 481-492 https://doi.org/10.1016/S0378-4320(00)00099-3
  24. Wells DN (2003): Cloning in livestock agriculture. Reprod Suppl 61:131-150
  25. Young LE, Fairburn HR (2000): Improving the safety of embryo technologies: Possible role of genomic imprinting. Theriogenology 53:627-648 https://doi.org/10.1016/S0093-691X(99)00263-0
  26. 박노형, 이성수, 정준, 원유석, 김내수 (2003): 한우 종모우의 고환둘레와 정액생산 및 번식과의 관계. 동물자원과학회지 45:517-522